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Designation: D 932 85 (Reapproved 2002)

An American National Standard

Standard Test Method for

Iron Bacteria in Water and Water-Formed Deposits1


This standard is issued under the fixed designation D 932; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.

5. Significance and Use


5.1 Iron bacteria is a general classification for microorganisms that utilize ferrous iron as a source of energy and are
characterized by the deposition of ferric hydroxide in their
mucilaginous sheaths. The process is continuous with these
growths, and over a period of time large accumulations of
slimey brown deposits can occur. Iron bacteria may clog water
lines, reduce heat transfer, and cause staining; objectionable
odors may arise following death of the bacteria. The organic
matter in the water is consequently increased, and this in turn
favors the multiplication of other bacteria.

1. Scope
1.1 This test method covers the determination of iron
bacteria by examination under the microscope. The method
provides for the identification of the following genera of
bacteria found in water and water-formed deposits: Siderocapsa, Gallionella (Dioymohelix), Sphaerotilus, Crenothrix,
Leptothrix, and Clonothrix.
1.2 This standard does not purport to address the safety
concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and
health practices and determine the applicability of regulatory
limitations prior to use.

6. Apparatus
6.1 Centrifuge, complete with conical tubes.
6.2 Microscope that provides a magnification of 400 to
10003 and is complete with a suitable light source. A
dark-field condenser is desirable.
6.3 Pipets, Mohr-type, 10-mL, with an opening 3 to 4 mm
in diameter, for thick samples, and 1-mL Mohr-type pipets for
thin samples.
6.4 Spatula, small and narrow, for handling thick samples.
6.5 Membrane Filter, with appropriate filter-holding assembly (see 9.2).

2. Referenced Documents
2.1 ASTM Standards:
D 887 Practices for Sampling Water-Formed Deposits2
D 1129 Terminology Relating to Water3
D 1193 Specification for Reagent Water3
D 3370 Practices for Sampling Water from Closed Conduits3
3. Terminology
3.1 DefinitionsFor definitions of terms used in this test
method, refer to Terminology D 1129.

7. Reagents
7.1 Purity of ReagentsReagent grade chemicals shall be
used in all tests. Unless otherwise indicated, it is intended that
all reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society,
where such specifications are available.5 Other grades may be
used, provided it is first ascertained that the reagent is of
sufficiently high purity to permit its use without lessening the
accuracy of the determination.
7.2 Purity of Water Unless otherwise indicated, references to water shall be understood to mean reagent water
conforming to Specification D 1193, Type II.
7.3 Ammonium Oxalate-Crystal Violet SolutionPrepare
Huckers modification of the Gram stain (4) by mixing a

4. Summary of Test Method


4.1 The iron bacteria are generally filamentous, typically
found in fresh water, and frequently surrounded by a sheath
which is usually encrusted with iron or manganese, or both (1,
2).4However, Starkey (3) reports another type which is classified among the true bacteria. Detection and identification is
accomplished by microscopical examination of sediment from
the sample. Table 1 and Figs. 1-10 (3) may be used to
differentiate the various types. This test method provides an
indication of the density of the iron bacteria and the severity of
the clogging problem in pipes caused by these bacteria.

1
This test method is under the jurisdiction of ASTM Committee D-19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved Aug. 30, 1985. Published October 1985. Originally
published as D 932 47 T. Last previous edition D 932 72 (1984)e 1.
2
Annual Book of ASTM Standards, Vol 11.02.
3
Annual Book of ASTM Standards, Vol 11.01.
4
The boldface numbers in parentheses refer to the list of references at the end of
this test method.

5
Reagent Chemicals, American Chemical Society Specifications, Am. Chemical Soc., Washington, DC. For suggestions on the testing of reagents not listed by
the American Chemical Society, see Reagent Chemicals and Standards, by Joseph
Rosin, D. Van Nostrand Co., Inc., New York, NY, and the United States
Pharmacopeia.

Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

D 932 85 (2002)
TABLE 1 Key for Identification of Bacteria

solution of 2.0 g of crystal violet (90 % dye content) in 20 mL


of ethyl alcohol (95 % with a solution of 0.8 g of ammonium
oxalate monohydrate (NH4)2C2O 4H2O) in 80 mL of water.
7.4 Hydrochloric Acid (1 + 4)Mix 1 volume of hydrochloric acid (HCl, sp gr 1.19) with 4 volumes of water.
7.5 Iodine Solution Prepare Grams modification of
Lugols solution (4) by dissolving 1 g of iodine in a solute
containing 2 g of potassium iodide (KI) in 10 mL of water and
diluting the resulting solution to 300 mL with water.
7.6 Filter Paper or Blotter.
7.7 Slides, standard type, 25 by 76-mm (1 by 3 in.) with
either plain or frosted end.
7.8 Cover Glasses, round or square type, 19 mm (34 in.) in
diameter.
8. Sampling
8.1 Collect the samples in accordance with either Practices
D 887 or D 3370, whichever is applicable.
8.2 Obtain a 500-mL (1-pt) sample of water, using a sterile
1-L (1-qt) bottle. The bottle should not be more than half-filled
because of the oxygen demand of suspended matter; filling the
bottle may cause the sample to become anaerobic.
8.3 If the number of iron bacteria are very low or that they
are just becoming established in the system, use a small side
stream filter to collect the sample to be examined. The water
suspected of containing iron bacteria should be filtered through
a highly retentive filter paper (or some other comparable
media) for 24 h. Centrifugation or membrane filtration is
satisfactory also. The flow rate of the water should be at the
maximum filtering capacity of the material employed.
8.4 Regardless of the method used to concentrate the solids
in the water, keep them moist until examined.

FIG. 1 Siderocapsa treubii. Multiple colonies surrounded by ferric


hydrate. Magnification about 500 3 . Fig. 4 of Ref (5)

D 932 85 (2002)

FIG. 3 Gallionella major. Curved cells at the ends of excretion


bands. Magnification about 1120 3 . Fig. 6 of Ref (6)

FIG. 2 Gallionella major. Cells at the ends of excretion bands


undergoing division. Magnification about 1180 3 . Fig. 3 of Ref
(6)

8.5 Mud samples should be collected from the mud-water


interface for maximum bacterial populations.
8.6 Transfer the deposit or mud samples to wide-mouth
bottles and add clean, chlorine-free water to cover the deposits
and maintain moisture until examined. Protect the samples
from sunlight and hold at 4C during transportation and
storage.
8.7 As soon as possible after collection of the solids,
microscopically examine them for the presence of iron bacteria.
9. Procedure
9.1 Place a portion of the sample on the slide and apply a
cover glass. A spatula or wide-mouth pipet can be used to
transfer the sample to the slide. Use a pipet when flocs of
material are encountered, as the flocs settle to the tip when the
pipet is held in a vertical position, and concentrate in the first
drop. In the case of very dilute solids or a water sample,
concentrate the organisms by centrifuging, pour off the supernatant liquid, and repeat if necessary.
9.2 An alternative procedure is to filter a suitable volume of
the dilute solids or the entire water sample through a 0.45-m
membrane filter in an appropriate membrane filtration assembly (holder, tubing, trap, flasks and vacuum pump). For this test

FIG. 4 Sphaerotilus dichotoma. Sketch showing false branching.


Magnification about 230 3 . Fig. 3b of Ref (7)

it is not necessary to sterilize the filter assembly for each


sample, but the assembly should be thoroughly cleaned between tests.
3

D 932 85 (2002)
9.4 Place the HCl (1 + 4) at one side of the cover glass and
draw it underneath by absorbing the liquid at the opposite side
by means of a filter paper or blotter. Continue this procedure
until no more yellow ferric chloride is evident in the solution.
Take care that the flow of the liquid is not fast, or the sample
may be drawn to the absorbent material. This treatment
removes the iron deposited in the sheaths of the bacteria and
allows the cells to be seen.
9.5 In a similar manner, rinse the iodine solution under the
cover glass until the color of the liquid becomes yellow or the
filter paper becomes colored. The iodine stains the bacterial
cells brown and makes them more easily visible.
9.6 Examine the slide under a microscope, using a highpower, dry objective, for the presence of Sphaerotilus, Crenothrix, Leptothrix, and Clonothrix. If used carefully, an oilimmersion lens may be helpful.
9.7 Prepare a new slide by placing a drop of the sample on
a clean slide and allowing it to air-dry. Then stain it for 1 min
with ammonium oxalate-crystal violet solution, wash it with
water, and allow it to dry. Examine the slide under anoilimmersion lens for the presence of Siderocapsa, which will
appear violet colored.
10. Report
10.1 The report shall state Present or Not found, probably absent. Make a statement as to the relative abundance of
the organisms present. Make a negative report only after
examination of several slides.

FIG. 5 Crenothrix polyspora. Sketch showing details of false


branching of cells within sheath. Magnification about 380 3 .
Plate 1, Fig. A of Ref (8)

11. Precision and Bias


11.1 Since this standard is a qualitative type test, precision
and bias statements cannot be provided.

9.3 Examine the slide under the microscope to determine if


encrusted or colorless sheaths are present. Note the presence of
the twisted stalks of Gallionella at this point, since treatment
with acid in accordance with 9.4 will dissolve the delicate
stalks.

D 932 85 (2002)

FIG. 6 Crenothrix polyspora. Cells enclosed within a sheath of ferric hydrate and showing false branching. Magnification about 390 3 .
Plate 3, Fig. B of Ref (8)

D 932 85 (2002)

FIG. 7 Leptothrix ochracea. Cells coming out of their sheath.


Magnification about 2200 3 . Plate 4, Fig. 20 of Ref (9)

FIG. 8 Leptothrix ochracea. Sheaths from an accumulation of


precipitated ferric hydrate in iron bearing water. Magnification
about 390 3 . Fig. 5 of Ref (7)

D 932 85 (2002)

FIG. 9 Clonothrix ferruginea. Sketch showing cells enclosed within sheath and
false branching. Magnification about 430 3 . Fig. 4 of Ref (7)

D 932 85 (2002)

FIG. 10 Crenothrix polyspora. Conidia can be seen inside and coming out at ends of filaments. Magnification about 345 3 . Fig. 5 of Ref
(9)

REFERENCES
(1) Bergey, D. H., Manual of Determinative Bacteriology, 8th Edition,
Williams & Wilkins Co., Baltimore, MD, 1974.
(2) Salle, A. J., Fundamental Principles of Bacteriology, McGraw-Hill
Book Co., Inc., New York, NY, 1943, pp. 516519.
(3) Starkey, R. L., Transformation of Iron by Bacteria in Water, Journal
of the American Water Works Association, Vol. 37, 1945, pp. 963984.
(4) Manual of Methods for Pure Culture Study of Bacteria, Biotech
Publications, Geneva, NY, 1946, Chapter IV, pp. 4648.

(5) Hardman, Yvette, and Henrici, A. T., Studies of Fresh Water Bacteria
V. Distribution of Siderocapsa treubii in Some Lakes and Streams,
Journal of Bacteriology, Vol 37, 1939, p. 97.
(6) Mitchell, R., Water Pollution Microbiology Vol. 1, Wiley-Interscience,
New York, NY, 1972.
(7) Standard Methods for the Examination of Water and Waste Water.
American Public Health Assoc., 19th Edition, 1976, p. 1000.

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